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In the present study we chose four cultures Bacillus licheniformis (MTCC 2617, MTCC 2618) and WILD A, WILD B (isolated from poultry waste soil), were screened for protease. In the production Bacillus licheniformis (MTCC 2617) and strain AR2 identified as Bacillus megaterium showed higher enzyme productivity (352U/ml and 321U/ml respectively). The enzymes were purified to its homogeneity by ammonium sulfate precipitation, dialysis and ion exchange chromatography. The characterization showed similarity in optimum pH (9) and optimum temperature (55°C). Serine protease from the different sources showed different molecular weight in SDS-PAGE. The amino acid sequences of Serine protease of Bacillus megaterium and Bacillus lichiniformis were downloaded from NCBI web server and aligned using ALIGN server's water program. It showed that there is 49% similarity between serine proteases produced from Bacillus licheniformis and Bacillus megaterium (AR2). From these data we concluded that production of serine protease can be done with Bacillus megaterium (AR2) an efficient and alternative to Bacillus licheniformis (MTCC 2617).